DNA supercoiling and thermal regulation of unsaturated fatty acid synthesis in Bacillus subtilis

Bacillus subtilis growing at 37° C synthesizes, almost exclusively, saturated fatty acids. However, when a culture growing at 37°C is transferred to 20°C, the synthesis of unsaturated fatty acids is induced. The addition of the DNA gyrase inhibitor novobiocin specifically prevented the induction of unsaturated fatty acid synthesis at 20° C. Furthermore, it was determined that plasmid DNA isolated from cells growing at 20°C was significantly more negatively supercoiled than the equivalent DNA isolated from cells growing at 37°C. The overall results agree with the hypothesis that an increase in DNA supercoiling associated with a temperature downshift could regulate the unsaturated fatty acids synthesis in B. subtilis.     

Binding of BiP to an assembly-defective protein in plant cells

The binding protein (BiP) has been implicated as a mediator of protein folding and assembly in the endoplasmic reticulum of mammalian cells and has often been found in stable association with structurally defective proteins. To acquire information on the activity of BiP in plant cells, we have expressed in tobacco protoplasts the wild type form and an assembly-defective form of bean phaseolin. Phaseolin (PHSL) is a soluble, trimeric, storage glycoprotein co-translationally inserted into the lumen of the endoplasmic reticulum and then transported along the secretory pathway to the protein storage vacuoles. We have previously shown that a PHSL mutant in which the last 59 amino acids have been deleted (Δ363PHSL) is unable to form trimers and is retained in a pre-Golgi compartment when synthesized in Xenopus oocytes. When transiently expressed in tobacco leaf protoplasts, wild-type PHSL is correctly glycosylated and assembles efficiently and rapidly into trimers. Δ363PHSL is also correctly glycosylated but does not trimerize. Tobacco BiP and Δ363PHSL are co-immunoselected using either anti-PHSL or anti-BiP antibodies. Under the same conditions, co-immunoselection of BiP with wild-type PHSL is not detectable. The BiP bound to Δ363PHSL can be released by treatment of the complex with ATP, indicating that the binding is related to the proposed function of BiP in protein folding and assembly in the endoplasmic reticulum. These data indicate that BiP stably binds structurally defective proteins in plant cells.     

Pigment production from in vitro cultures of Alkanna tinctoria Tausch

Summary An in vitro culture of Alkanna tinctoria Tausch cells was set up in order to investigate the possibility of producing alkannin, a red naphthoquinone naturally present in the root bark of this plant. Furthermore, an in vitro culture of callusderived roots was established and the production of alkannin evaluated. In the different experimental conditions investigated, differences in the production of alkannin derivatives as well as in the type of pigments produced, were observed. The potential use of this technology is discussed.     

Cooperative effect of inositol hexakisphosphate, bezafibrate, and clofibric acid on the spectroscopic properties of the nitric oxide derivative of ferrous human hemoglobin

The cooperative effect of inositol hexakisphosphate (IHP), bezafibrate (BZF), and clofibric acid (CFA) on the spectroscopic (EPR and absorbance) properties of the nitric oxide derivative of ferrous human hemoglobin (HbNO) has been investigated quantitatively. In the presence of IHP, BZF, and CFA, the X-band EPR spectra and the absorption spectra in the Soret region of HbNO display the same basic characteristics described in the presence of 2,3-diphosphoglycerate (2,3-DPG), which have been attributed to a low affinity conformation of the tetramer. Addition to HbNO of two allosteric effectors together (such as IHP and BZF, or IHP and CFA) further stabilizes the low affinity conformation of the ligated hemoprotein (i.e., HbNO). Moreover, in the presence of saturating amounts of IHP, the affinity of BZF and CFA for HbNO increases by about fifteenfold. Likewise, in the presence of both IHP and BZF, as well as in IHP and CFA, the oxygen affinity for ferrous human hemoglobin (Hb) is reduced with respect to that observed in the presence of IHP, BZF, or CFA alone, which in turn is lower than that reported in the absence of any allosteric effector. All the data were obtained at pH 7.0 (in 1.0 × 10⁻¹ M N-[2-hydroxyethyl]piperazine-N′-[2-ethanesulfonic acid]/NaOH buffer system plus 1.0 × 10⁻¹ M NaCl), as well as at 100 K and/or 20°C. The results here reported represent clearcut evidence for the cooperative and specific (i.e., functionally relevant) binding of IHP, BZF, and CFA to Hb.     

Thermostable β-Glycosidase from Sulfolobus Solfataricus

The Sulfolobus solfataricus β-glycosidase (Sβgly) is a thermostable and thermophilic glycosyl-hydrolase with broad substrate specificity. The enzyme hydrolizes β-D-gluco-, fuco-, and galactosides, and a large number of /Winked glycoside dimers and oligomers, linked β1-3, β1-4, and β1-6, It is able to hydrolize oligosaccharides with up to 5 glucose residues. Furthermore, it is also able to promote transglycosylation reactions. The corresponding gene has been cloned and overexpressed both in yeast and Escherichia coli. Based on sequence and functional data, the Sβgly has been assigned to the so-called BGA family of glycosyl-hydrolases, including β-glycosidases, β-galactosidases and phosho-β-galactosidases from mesophilic and thermophilic organisms of the three domains. The Sβgly has been crystallized and the resolution of its structure is in progress. Because of its special properties, the enzymes has considerable biotechnological potential.     

Human hypertensive placenta contains an increased amount of Na,K-ATPase with higher affinity for cardiac glycosides

Placentas of women suffering from pregnancy-induced hypertension (PIH) were found to contain a greater amount of Na,K-ATPase molecules, estimated from anthroyl ouabain binding, than normotensive individuals. Both the microsomal fraction of placental cells and purified Na,K-ATPase showed an increased affinity for the specific inhibitor ouabain which, in the case of the microsomes, bound with a dissociation constant of 0.9 nM as compared with 3.4 nM in the controls. Likewise, the dissociation constant of the ouabain complex with purified Na,K-ATPase was about 3.5 times lower in the hypertensive patients. The differences are apparently caused by a different microenvironment of the ouabain-binding site, as reflected in the quantum yield of bound anthroyl ouabain. If an endogenous digitalis-like factor is present in the body fluids to regulate Na,K-ATPase activity, the present results render its role quite plausible.     

Characterization of hemocytes from the marine amphipod Parhyale hawaiensis (Dana 1853): Setting the basis for immunotoxicological studies

Hemocytes are circulating blood cells that play a crucial function in amphipods and other crustacean immune systems. The hemocytes of the marine tropical amphipod Parhyale hawaiensis have been used for the evaluation of DNA damage and micronuclei, but they have not been characterized in the scientific literature. The aim of this study was to describe the hemolymph cells of P. hawaiensis and study their phagocytotic activity. Basic dyes were used to differentiate the cell types and the presence of lipids. The total hemocyte counts (THCs) and the proportion and sizes of the hemocyte types were determined. Hemolymph was exposed to Escherichia coli for verification of the presence of phagocytosis. Three cell types, all containing lipids, were identified in P. hawaiensis: granulocytes (oval shape, 13.4 × 7.6 μm), semi-granulocytes (oval shape, 14.1 × 7.2 μm), and hyalinocytes (round shape, 9.6 × 7.2 μm). Those three cell types were found in different percentages in males (64.8%, 31.1%, and 4.2%) and females (70.1%, 28.2%, and 1.7%). THCs for males were 9007 ± 3800 cells per individual and 4695 ± 1892 cells per individual for females. The cells of E. coli were phagocytized by the hemocytes. Our findings increased the knowledge of hemocytes in P. hawaiensis and is a step forward in using hemocyte-based immune responses as an endpoint in ecotoxicology.     

Erk1/2 signaling mediates the HGF-induced protection against ethanol and acetaldehyde-induced toxicity in the pancreatic RINm5F cell line

Alcohol-induced pancreas damage remains as one of the main risk factors for pancreatitis development. This disorder is poorly understood, particularly the effect of acetaldehyde, the primary alcohol metabolite, in the endocrine pancreas. Hepatocyte growth factor (HGF) is a protective protein in many tissues, displaying antioxidant, antiapoptotic, and proliferative responses. In the present work, we were focused on characterizing the response induced by HGF and its protective mechanism in the RINm5F pancreatic cell line treated with ethanol and acetaldehyde. RINm5F cells were treated with ethanol or acetaldehyde for 12 h in the presence or not of HGF (50 ng/ml). Cells under HGF treatment decreased the content of reactive oxygen species and lipid peroxidation induced by both toxics, improving cell viability. This effect was correlated to an improvement in insulin expression impaired by ethanol and acetaldehyde. Using a specific inhibitor of Erk1/2 abrogated the effects elicited by the growth factor. In conclusion, the work provides mechanistic evidence of the HGF-induced-protective response to the alcohol-induced damage in the main cellular component of the endocrine pancreas.     

Smart hydrogels based on semi-interpenetrating polymeric networks of collagen-polyurethane-alginate for soft/hard tissue healing,drug delivery devices,and anticancer therapies

In this work, hydrogels based on semi-interpenetrating polymeric networks (semi-IPN) based on collagen-polyurethane-alginate were studied physicochemically and from different approaches for biomedical application. It was determined that the matrices in the hydrogel state are crosslinked by the formation of urea and amide bonds between the biopolymer chains and the polyurethane crosslinker. The increment in alginate content (0–40 wt%) significantly increases the swelling capacity, generating semi-crystalline granular structures with improved storage modulus and resistance to thermal, hydrolytic, and proteolytic degradation. The in vitro bioactivity results indicated that the composition of these novel hydrogels stimulates the metabolic activity of monocytes and fibroblasts, benefiting their proliferation; while in cancer cell lines, it was determined that the composition of these biomaterials decreases the metabolic activity of breast cancer cells after 48 h of stimulation, and for colon cancer cells their metabolic activity decreases after 72 h of contact for the hydrogel with 40 wt% alginate. The matrices show a behavior of multidose release of ketorolac, and a higher concentration of analgesic is released in the semi-IPN matrix. The inhibition capacity of Escherichia coli is higher if the polysaccharide concentration is low (10 wt%). The in vitro wound closure test (scratch test) results indicate that the hydrogel with 20 wt% alginate shows an improvement in wound closure at 15 days of contact. Finally, the bioactivity of mineralization was evaluated to demonstrate that these hydrogels can induce the formation of carbonated apatite on their surface. The engineered hydrogels show biomedical multifunctionality and they could be applied in soft and hard tissue healing strategies, anticancer therapies, and drug release devices.